Inhibition of transforming growth factor beta activity

ABSTRACT

The present invention provides a method of inhibiting an activity of TGFβ comprising contacting the TGFβ with a purified decorin. In a specific embodiment, the present invention relates to the ability of decorin, a 40,000 dalton protein that usually carries a glycosaminoglycan chain, to bind TGFβ. The invention also provides a novel cell regulatory factor designated MRF. Also provided are methods of identifying, detecting and purifying cell regulatory factors and proteins which bind and affect the activity of cell regulatory factors.

This invention was made with support of government grants CA 30199, CA42507 and CA 28896 from the National Cancer Institute. Therefore, theUnited States government may have rights in the invention.

This application is a continuation of application Ser. No. 08/050,762,filed Apr. 20, 1993, now abandoned, which is a continuation ofapplication Ser. No. 07/467,888, filed on Jan. 22, 1990, now abandoned,which is a continuation of application Ser. No. 07/212,702, filed Jun.28, 1988 now abandoned.

FIELD OF INVENTION

This invention relates to cell biology and more specifically to thecontrol of cell proliferation.

BACKGROUND OF THE INVENTION

Proteoglycans are proteins that carry one or more glycosaminoglycanchains. The known proteoglycans carry out a wide variety of functionsand are found in a variety of cellular locations. Many proteoglycans arecomponents of extracellular matrix, where they participate in theassembly of cells and effect the attachment of cells to the matrix.

Decorin, also known as PG-II or PG-40, is a small proteoglycan producedby fibroblasts. Its core protein has a molecular weight of about 40,000daltons. The core has been sequenced (Krusius and Ruoslahti, Proc. Natl.Acad. Sci. USA 83:7683 (1986); Day et al. Biochem. J. 248:801 (1987),both of which are incorporated herein by reference) and it is known tocarry a single glycosaminoglycan chain of a chondroitin sulfate/dermatansulfate type (Pearson, et al., J. Biol. Chem. 258:15101 (1983), which isincorporated herein by reference). The only previously known functionfor decorin is binding to type I and type II collagen and its effect onthe fibril formation by these collagens (Vogel, et al., Biochem. J.223:587 (1984); Schmidt et al., J. Cell Biol. 104:1683, (1987)). Twoproteoglycans, biglycan (Fisher et al., J. Biol. Chem. 264:4571 (1989))and fibromodulin, (Oldberg et al., Embo J. 8:2601, (1989) have coreproteins the amino acid sequences of which are closely related to thatof decorin and they, together with decorin, can be considered a proteinfamily. Each of their sequences is characterized by the presence of aleucine-rich repeat of about 24 amino acids. Several other proteinscontain similar repeats. Together all these proteins form a superfamilyof proteins (Ruoslahti, Ann. Rev. Cell Biol. 4:229, (1988); McFarland etal., Science 245:494 (1989)).

Transforming growth factor β's (TGFβ) are a family of multifunctionalcell regulatory factors produced in various forms by many types of cells(for review see Sporn et al., J. Cell Biol. 105:1039, (1987)). Fivedifferent TGFβ's are known, but the functions of only two, TGFβ-1 andTGFβ-2, have been characterized in any detail. TGFβ's are the subject ofU.S. Pat. Nos. 4,863,899; 4,816,561; and 4,742,003 which areincorporated by reference. TGFβ-1 and TGFβ-2 are publicly availablethrough many commercial sources (e.g. R & D Systems, Inc., Minneapolis,Minn.). These two proteins have similar functions and will be herecollectively referred to as TGFβ. TGFβ binds to cell surface receptorspossessed by essentially all types of cells, causing profound changes inthem. In some cells, TGFβ promotes cell proliferation, in others itsuppresses proliferation. A marked effect of TGFβ is that it promotesthe production of extracellular matrix proteins and their receptors bycells (for review see Keski-Oja et al., J. Cell Biochem 33:95 (1987);Massague, Cell 49:437 (1987); Roberts and Sporn in "Peptides GrowthFactors and Their Receptors" [Springer-Verlag, Heidelberg] in press(1989)).

While TGFβ has many essential cell regulatory functions, improper TGFβactivity can be detrimental to an organism. Since the growth ofmesenchyme and proliferation of mesenchymal cells is stimulated by TGFβ,some tumor cells may use TGFβ as an autocrine growth factor. Therefore,if the growth factor activity of TGFβ could be prevented, tumor growthcould be controlled. In other cases the inhibition of cell proliferationby TGFβ may be detrimental, in that it may prevent healing of injuredtissues. The stimulation of extracellular matrix production by TGFβ isimportant in situations such as wound healing. However, in some casesthe body takes this response too far and an excessive accumulation ofextracellular matrix ensues. An example of excessive accumulation ofextracellular matrix is glomerulonephritis, a disease with a detrimentalinvolvement of TGFβ.

Thus, there exists a critical need to develop compounds that canmodulate the effects of cell regulatory factors such as TGFβ. Thepresent invention satisfies this need and provides related advantages.

SUMMARY OF THE INVENTION

The present invention provides a method of inhibiting an activity of acell regulatory factor comprising contacting the cell regulatory factorwith a purified polypeptide, wherein the polypeptide comprises a cellregulatory factor binding domain of a protein and wherein the protein ischaracterized by a leucine-rich repeat of about 24 amino acids. In aspecific embodiment, the present invention relates to the ability ofdecorin, a 40,000 dalton protein that usually carries aglycosaminoglycan chain, to bind TGFβ. The invention also provides anovel cell regulatory factor designated Morphology Restoring Factor,(MRF). Also provided are methods of identifying, detecting and purifyingcell regulatory factors and proteins which bind and affect the activityof cell regulatory factors.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show expression of decorin cDNA containing a mutation ofthe serine acceptor site to alanine. COS-1 cultures were transfectedwith cDNA coding for wild-type decorin (lane 1), decorin in which theserine-4 residue was replaced by an alanine (lane 2), or decorin inwhich the serine-4 residue was replaced by a threonine (lane 3).Immunoprecipitations were performed with an anti-decorin antibody andmedium which was labeled with ³⁵ S-sulfate (FIG. 1A) or ³ H-leucine(FIG. 1B). Lane 4 shows an immunoprecipitate from mock transfected COS-1cultures. Arrow indicates top of gel. The numbers indicate M_(r) ×10⁻³for molecular weight standards.

FIGS. 2A and 2B show binding of [¹²⁵ I]TGFβ1 to decorin-Sepharose.

(FIG. 2A) Fractionation of [¹²⁵ I]-TGFβ1 by decorin-Sepharose affinitychromatography. [¹²⁵ I]TGFβ1 (5×10⁵ cpm) was incubated in BSA-coatedpolypropylene tubes with 0.2 ml of packed decorin-Sepharose () orgelatin-Sepharose (0) in 2 ml of PBS pH 7.4, containing 1M NaCl and0.05% Tween 20. After overnight incubation, the affinity matrices weretransferred into BSA-coated disposable columns (Bio Rad) and washed withthe binding buffer. Elution was effected first with 3M NaCl in thebinding buffer and then with 8M urea in the same buffer.

(FIG. 2B) Analysis of eluents of decorin-Sepharose affinitychromatography by SDS-polyacrylamide gel under nonreducing conditions.Lane 1: the original [¹²⁵ I]-labeled TGFβ1 sample; lanes 2-7: flowthrough and wash fractions; lanes 8-10: 3M NaCl fractions; lanes 11-14:8M urea fractions. Arrows indicate the top and bottom of the 12%separating gel.

FIGS. 3A and 3B show the inhibition of binding of [¹²⁵ I]TGFβ1 todecorin by proteoglycans and their core proteins.

(FIG. 3A) Competition of [¹²⁵ I]TGFβ1 binding to decorin-coatedmicrotiter wells by recombinant decorin (), decorin isolated frombovine skin (PGII) (▪), biglycan isolated from bovine articularcartilage (PGI) (▴), chicken cartilage proteoglycan (∘), and BSA (□).Each point represents the mean of duplicate determinants.

(FIG. 3B) Competition of [¹²⁵ I]TGFβ1 binding with chondroitinaseABC-treated proteoglycans and BSA. The concentrations of competitorswere expressed as intact proteoglycan. The symbols are the same as inFIG. 3A.

FIGS. 4A and 4B show neutralization of the growth regulating activity ofTGFβ1 by decorin.

(FIG. 4A) Shows inhibition of TGFβ1-induced proliferation of CHO cellsby decorin. [³ H]Thymidine incorporation assay was performed asdescribed in the legend of FIG. 1 in the presence of 5 ng/ml of TGFβ-1and the indicated concentrations of purified decorin () or BSA (0). Atthe concentration used, TGFβ-1 induced a 50% increase of [³ H]thymidineincorporation in the CHO cells. The data represent percentneutralization of this growth stimulation; i.e. [³ H]thymidineincorporation in the absence of either TGFβ1 or decorin=0%,incorporation in the presence of TGFβ but not decorin=100%. Each pointshows the mean±standard deviation of triplicate samples.

(FIG. 4B) Shows neutralization of TGFβ1-induced growth inhibition inMv1Lu cells by decorin. Assay was performed as in A except that TGFβ-1was added at 0.5 ng/ml. This concentration of TGFβ-1 induces 50%reduction of [³ H]thymidine incorporation in the Mv1Lu cells. The datarepresent neutralization of TGFβ-induced growth inhibition; i.e. [³H]thymidine incorporation in the presence of neither TGFβ ordecorin=100%; incorporation in the presence of TGFβ but not decorin=0%.

FIG. 5A shows separation of growth inhibitory activity fromdecorin-expressing CHO cells by gel filtration. Serum-free conditionedmedium of decorin overexpressor cells was fractionated by DEAE-Sepharosechromatography in a neutral Tris-HCl buffer and fractions containinggrowth inhibitory activity were pooled, made 4M with guanidine-HCl andfractionated on a Sepharose CL-6B column equilibrated with the sameguanidine-HCl solution. The fractions were analyzed for protein content,decorin content, and growth regulatory activities. Elution positions ofmarker proteins are indicated by arrows. BSA: bovine serum albumin(Mr=66,000); CA: carbonic anhydrase (Mr=29,000); Cy: cytochrome c(Mr=12,400); Ap: aprotinin (Mr=6,500); TGF: [¹²⁵ I]TGFβ1 (Mr=25,000).

FIG. 5B shows identification of the growth stimulatory material from gelfiltration as TGFβ1. The growth stimulatory activity from the latefractions from Sepharose 6B (bar in FIG. 5A) was identified byinhibiting the activity with protein A-purified IgG from an anti-TGFβantiserum. Data represent percent inhibition of growth stimulatoryactivity in a [³ H]thymidine incorporation assay. Each point shows themean ±standard deviation of triplicate determinations. Anti-TGFβ1 (),normal rabbit IgG (◯).

FIGS. 6A and 6B show micrographs demonstrating a decorin-binding cellregulatory activity that is not suppressed by antibodies to TGFβ-1.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of inhibiting an activity of a cellregulatory factor comprising contacting the cell regulatory factor witha purified polypeptide, wherein the polypeptide comprises the cellregulatory factor binding domain of a protein and wherein the protein ischaracterized by a leucine-rich repeat of about 24 amino acids. Sincediseases such as cancer result from uncontrolled cell proliferation, theinvention can be used to treat such diseases.

By "cell regulatory factor" is meant a molecule which can regulate anactivity of a cell. The cell regulatory factors are generally proteinswhich bind cell surface receptors and include growth factors. Examplesof cell regulatory factors include the five TGFβ's, platelet-derivedgrowth factor, epidermal growth factor, insulin like growth factor I andII, fibroblast growth factor, interleukin-2, nerve growth factor,hemopoietic cell growth factors (IL-3, GM-CSF, M-CSF, G-CSF,erythropoietin) and the newly discovered Morphology Restoring Factor,hereinafter "MRF". Different regulatory factors can be bound bydifferent proteins which can affect the regulatory factor's activity.For example, TGFβ-1 is bound by decorin and biglycan, and MRF bydecorin.

By "cell regulatory factor binding domain" is meant the fragment of aprotein which binds to the cell regulatory factor. While the specificexamples set forth herein utilize proteins, it is understood that aprotein fragment which retains the binding activity is included withinthe scope of the invention. Fragments which retain such activity can berecognized by their ability to competitively inhibit the binding of, forexample, decorin to TGFβ, or of other polypeptides containingleucine-rich repeats to their cognate growth factors. As an example,fragments can be obtained by digestion of the native polypeptide or bysynthesis of fragments based on the known amino acid sequence. Suchfragments can then be used in a competitive assay to determine whetherthey retain binding activity. For example, decorin can be attached to anaffinity matrix, as by the method of Example II. Labelled TGFβ, and thefragment in question can then be contacted with the affinity matrix andthe amount of TGFβ bound thereto determined.

As used herein, "decorin" refers to a proteoglycan having substantiallythe structural characteristics attributed to it in Krusius andRuoslahti, supra. Human fibroblast decorin has substantially the aminoacid sequence presented in Krusius and Ruoslahti, supra. "Decorin"refers both to the native composition and to modifications thereof whichsubstantially retain the functional characteristics. Decorin coreprotein refers to decorin that no longer is substantially substitutedwith glycosaminoglycan and is included in the definition of decorin.Decorin can be rendered glycosaminoglycan-free by mutation or othermeans, such as by producing recombinant decorin in cells incapable ofattaching glycosaminoglycan chains to a core protein.

Since the regulatory factor binding proteins each contain leucine-richrepeats of about 24 amino acids which can constitute 80% of the protein,it is likely that the fragments which retain the binding activity occurin the leucine-rich repeats. However, it is possible the bindingactivity resides in the carboxy terminal amino acids or the junction ofthe repeats and the carboxy terminal amino acids.

The invention teaches a general method whereby one skilled in the artcan identify proteins which can bind to cell regulatory factors oridentify cell regulatory factors which bind to a certain family ofproteins. The invention also teaches a general method whereby thesenovel proteins or known existing proteins can be assayed to determine ifthey affect an activity of a cell regulatory factor. Specifically, theinvention teaches the discovery that decorin and biglycan bind TGFβ-1and MRF and that such binding can inhibit the cell regulatory functionsof TGFβ-1. Further, both decorin and biglycan are about 80% homologousand contain a leucine-rich repeat of about 24 amino acids in which thearrangement of the leucine residues is conserved. As defined each repeatgenerally contains at least two leucine residues and can contain five ormore. These proteoglycans are thus considered members of the sameprotein family. See Ruoslahti, supra, Fisher et al., J. Biol. Chem.,264:4571-4576 (1989) and Patthy, J. Mol. Biol., 198:567-577 (1987), allof which are incorporated by reference. Other known or later discoveredproteins having this leucine-rich repeat, i.e., fibromodulin, would beexpected to have a similar cell regulatory activity. The ability of suchproteins to bind cell regulatory factors could easily be tested, forexample by affinity chromatography or microtiter assay as set forth inExample II, using known cell regulatory factors, such as TGFβ-1.Alternatively, any later discovered cell regulatory factor could betested, for example by affinity chromatography using one or moreregulatory factor binding proteins. Once it is determined that suchbinding occurs, the effect of the binding on the activity of allregulatory factors can be determined by methods such as growth assays asset forth in Example III. Moreover, one skilled in the art could simplysubstitute a novel cell regulatory factor for TGFβ-1 or a novelleucine-rich repeat protein for decorin or biglycan in the Examples todetermine their activities. Thus, the invention provides general methodsto identify and test novel cell regulatory factors and proteins whichaffect the activity of these factors.

The invention also provides a novel purified compound comprising a cellregulatory factor attached to a purified polypeptide wherein thepolypeptide comprises the cell regulatory factor binding domain of aprotein and the protein is characterized by a leucine-rich repeat ofabout 24 amino acids.

The invention further provides a novel purified protein, designated MRF,having a molecular weight of about 20 kd, which can be isolated from CHOcells, copurifies with decorin under nondissociating conditions,separates from decorin under dissociating conditions, changes themorphology of transformed 3T3 cells, and has an activity which is notinhibited with anti-TGFβ-1 antibody. Additionally, MRF separates fromTGFβ-1 in HPLC.

The invention still further provides a method of purifying a cellregulatory factor comprising contacting the regulatory factor with aprotein which binds the cell regulatory factor and has a leucine-richrepeat of about 24 amino acids and to purify the regulatory factor whichbecomes bound to the protein. The method can be used, for example, topurify TGFβ-1 by using decorin.

The invention additionally provides a method of treating a pathologycaused by a TGFβ-regulated activity comprising contacting the TGFβ witha purified polypeptide, wherein the polypeptide comprises the TGFβbinding domain of a protein and wherein the protein is characterized bya leucine-rich repeat of about 24 amino acids, whereby thepathology-causing activity is prevented or reduced. While the method isgenerally applicable, specific examples of pathologies which can betreated include a cancer, a fibrotic disease, and glomerulonephritis. Incancer, for example, decorin can be used to bind TGFβ-1, destroyingTGFβ-1's growth stimulating activity on the cancer cell.

Finally, a method of preventing the inhibition of a cell regulatoryfactor is provided. The method comprises contacting a protein whichinhibits an activity of a cell regulator factor with a molecule whichinhibits the activity of the protein. For example, decorin could bebound by a molecule, such as an antibody, which prevents decorin frombinding TGFβ-1, thus preventing decorin from inhibiting the TGFβ-1activity. Thus, the TGFβ-1 wound healing activity could be promoted bybinding TGFβ-1 inhibitors.

It is understood that modifications which do not substantially affectthe activity of the various molecules of this invention including TGFβ,MRF, decorin, biglycan and fibromodulin are also included within thedefinition of those molecules. It is also understood that the coreproteins of decorin, biglycan and fibromodulin are also included withinthe definition of those molecules.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE I Expression and Purification of Recombinant Decorin and DecorinCore Protein

Expression System

The 1.8 kb full-length decorin cDNA described in Krusius and Ruoslahti,Proc. Natl. Acad. Sci. USA 83:7683 (1986), which is incorporated hereinby reference, was used for the construction of decorin expressionvectors. For the expression of decorin core protein, cDNA wasmutagenized so the fourth codon, TCT, coding for serine, was changed toACT coding for threonine, or GCT coding for alanine. This was engineeredby site-directed mutagenesis according to the method of Kunkel, Proc.Natl. Acad. Sci USA 82:488 (1985), which is incorporated herein byreference. The presence of the appropriate mutation was verified by DNAsequencing.

The mammalian expression vectors pSV2-decorin and pSV2-decorin/CP-thr4core protein were constructed by ligating the decorin cDNA or themutagenized decorin cDNA into 3.4 kb HindIII-Bam HI fragment of pSV2(Mulligan and Berg, Science 209:1423 (1980), which is incorporatedherein by reference).

Dihydrofolate reductase (dhfr)-negative CHO cells (CHO-DG44) werecotransfected with pSV2-decorin or pSV2-decorin/CP and pSV2dhfr by thecalcium phosphate coprecipitation method. The CHO-DG44 cells transfectedwith pSV2-decorin are deposited with the American Type CultureCollection under Accession Number CRL 10332. The transfected cells werecultured in nucleoside-minus alpha-modified minimal essential medium(α-MEM), (GIBCO, Long Island) supplemented with 9% dialyzed fetal calfserum, 2 mM glutamine, 100 units/ml penicillin and 100 μg/mlstreptomycin. Colonies arising from transfected cells were picked usingcloning cylinders, expanded and checked for the expression of decorin byimmunoprecipitation from ³⁵ SO₄ -labeled culture supernatants. Clonesexpressing a substantial amount of decorin were then subjected to geneamplification by stepwise increasing concentration of methotrexate (MTX)up to 0.64 μM (Kaufman and Sharp, J. Mol. Biol. 159:601 (1982), which isincorporated herein by reference). All the amplified cell lines werecloned either by limiting dilution or by picking single MTX resistantcolonies. Stock cultures of these established cell lines were kept inMTX-containing medium. Before use in protein production, cells weresubcultured in MTX-minus medium from stock cultures and passed at leastonce in this medium to eliminate the possible MTX effects.

Alternatively, the core protein was expressed in COS-1 cells asdescribed in Adams and Rose, Cell 41:1007, (1985), which is incorporatedherein by reference. Briefly, 6-well multiwell plates were seeded with3-5×10⁵ cells per 9.6 cm² growth area and allowed to attach and grow for24 hours. Cultures were transfected with plasmid DNA when they were50-70% confluent. Cell layers were washed briefly with Tris bufferedsaline (TBS) containing 50 mM Tris, 150 mM NaCl pH 7.2, supplementedwith 1 mM CaCl₂ and 0.5 mM MgCl₂ at 37° C. to prevent detachment. Thewells were incubated for 30 minutes at 37° C. with 1 ml of the abovesolution containing 2 μg of closed circular plasmid DNA and 0.5 mg/mlDEAE-Dextran (Sigma) of average molecular mass of 500,000. As a control,cultures were transfected with the pSV2 expression plasmid lacking anydecorin insert or mock transfected with no DNA. Culture were thenincubated for 3 hours at 37° C. with Dulbecco's Modified Eagle's medium(Irvine Scientific) containing 10% fetal calf serum and 100 μMchloroquine (Sigma), after removing the DNA/TBS/DEAE-Dextran solutionand rinsing the wells with TBS. The cell layers were then rinsed twiceand cultured in the above medium, lacking any chloroquine, forapproximately 36 hours. WI38 human embryonic lung fibroblasts wereroutinely cultured in the same medium.

COS-1 cultures were radiolabeled 36-48 hours after transfection with theplasmid DNAs. All radiolabeled metabolic precursors were purchased fromNew England Nuclear (Boston, Mass.). The isotopes used were ³⁵ S-sulfate(460 mCi/ml), L-[3,4,5-³ H(N)]--leucine (140 Ci/ml) and L-[¹⁴C(U)]--amino acid mixture (product number 445E). Cultures were labeledfor 24 hours in Ham's F-12 medium (GIBCO Labs), supplemented with 10%dialyzed fetal calf serum, 2 mM glutamine and 1 mM pyruvic acid, andcontaining 200 μCi/ml ³⁵ S-sulfate or ³ H-leucine, or 10 μCi/ml of the¹⁴ C-amino acid mixture. The medium was collected, supplemented with 5mM EDTA, 0.5 mM phenylmethylsulfonylfluoride, 0.04 mg/ml aprotinin and 1μg/ml pepstatin to inhibit protease activity, freed of cellular debrisby centrifugation for 20 minutes at 2,000×G and stored at -20° C. Cellextracts were prepared by rinsing the cell layers with TBS and thenscraping with a rubber policeman into 1 ml/well of ice cold cell lysisbuffer: 0.05M Tris-HCl, 0.5M NaCl, 0.1% BSA, 1% NP-40, 0.5% TritonX-100, 0.1% SDS, pH 8.3. The cell extracts were clarified bycentrifugation for 1.5 hours at 13,000×G at 4° C.

Rabbit antiserum was prepared against a synthetic peptide based on thefirst 15 residues of the mature form of the human decorin core protein(Asp-Glu-Ala-Ser-Gly-Ile-Gly-Pro-Glu-Val-Pro-Asp-Asp-Arg-Asp). Thesynthetic peptide and the antiserum against it have been describedelsewhere (Krusius and Ruoslahti, 1986 Supra.) Briefly, the peptide wassynthesized with a solid phase peptide synthesizer (Applied Biosystems,Foster City, Calif.) by using the chemistry suggested by themanufacturer. The peptide was coupled to keyhole limpet hemocyanin byusing N-succinimidyl 3-(2-pyridyldithio) propionate (Pharmacia FineChemicals, Piscataway, N.J.) according to the manufacturer'sinstructions. The resulting conjugates were emulsified in Freund'scomplete adjuvant and injected into rabbits. Further injections ofconjugate in Freund's incomplete adjuvant were given after one, two andthree months. The dose of each injection was equivalent to 0.6 mg ofpeptide. Blood was collected 10 days after the third and fourthinjection. The antisera were tested against the glutaraldehyde-crosslinked peptides and isolated decorin in ELISA (Engvall, Meth. Enzymol.70:419-439 (1980)), in immunoprecipitation and immunoblotting, and bystaining cells in immunofluorescence, as is well known in the art.

Immunoprecipitations were performed by adding 20 μl of antiserum to theconditioned medium or cell extract collected from duplicate wells andthen mixing overnight at 4° C. Immunocomplexes were isolated byincubations for 2 hours at 4° C. with 20 μl of packed Protein A-agarose(Sigma). The beads were washed with the cell lysis buffer, with threetube changes, and then washed twice with phosphate-buffered saline priorto boiling in gel electrophoresis sample buffer containing 10%mercaptoethanol. Immunoprecipitated proteins were separated by SDS-PAGEin 7.5-20% gradient gels or 7.5% non-gradient gels as is well known inthe art. Fluorography was performed by using Enlightning (New EnglandNuclear) with intensification screens. Typical exposure times were for7-10 days at -70° C. Autoradiographs were scanned with an LKB UltroscanXL Enhanced Laser Densitometer to compare the relative intensities andmobilities of the proteoglycan bands.

SDS-PAGE analysis of cell extracts and culture medium from COS-1 cellstransfected with the decorin-pSV2 construct and metabolicallyradiolabeled with ³⁵ S-sulfate revealed a sulfated band that was notpresent in mock-transfected cells. Immunoprecipitation with theantiserum raised against a synthetic peptide derived from the decorincore protein showed that the new band was decorin.

Expression of the construct mutated such that the serine residue whichis normally substituted with a glycosaminoglycan (serine-4) was replacedby a threonine residue by SDS-PAGE revealed only about 10% of the levelof proteoglycan obtained with the wild-type construct. The rest of theimmunoreactive material migrated at the position of free core protein.

The alanine-mutated cDNA construct when expressed and analyzed in asimilar manner yielded only core protein and no proteoglycan form ofdecorin. FIGS. 1A and B show the expression of decorin (lanes 1) and itsthreonine-4 (lanes 3) and alanine-4 (lanes 2) mutated core proteinsexpressed in COS cell transfectants. ³⁵ SO₄ -labeled (FIG. 1A) and ³H-leucine labeled (FIG. 1B) culture supernatants were immunoprecipitatedwith rabbit antipeptide antiserum prepared against the NH₂ -terminus ofhuman decorin.

Purification of Decorin and Decorin Core Protein from Spent CultureMedia

Cells transfected with pSV2-decorin vector and amplified as describedabove and in Yamaguchi and Ruoslahti, Nature 36:244246 (1988), which isincorporated herein by reference, were grown to 90% confluence in 8 175cm² culture flasks in nucleoside minus α-MEM supplemented with 9%dialyzed fetal calf serum, 2 mM glutamine, 100 units/ml penicillin and100 μg/ml streptomycin. At 90% confluence culture media was changed to25 ml per flask of nucleoside-free α-MEM supplemented with 6% dialyzedfetal calf serum which had been passed through a DEAE Sepharose FastFlow column (Pharmacia) equilibrated with 0.25M NaCl in 0.05M phosphatebuffer, pH 7.4. Cells were cultured for 3 days, spent media wascollected and immediately made to 0.5 mM phenylmethylsulfonyl fluoride,1 μg/ml pepstatin, 0.04 mg/ml aprotinin and 5 mM EDTA.

Four hundred milliliters of the spent media were first passed throughgelatin-Sepharose to remove fibronectin and materials which would bindto Sepharose. The flow-through fraction was then mixed withDEAE-Sepharose pre-equilibrated in 50 mM Tris/HCl, pH 7.4, plus 0.2MNaCl and batch absorbed overnight at 4° C. with gentle mixing. Theslurry was poured into a 1.6×24 cm column, washed extensively with 50mMTris/HCl, pH 7.4, containing 0.2M NaCl and eluted with 0.2M-0.8Mlinear gradient of NaCl in 50 mM Tris/HCl, pH 7.4. Decorin concentrationwas determined by competitive ELISA as described in Yamaguchi andRuoslahti, supra. The fractions containing decorin were pooled andfurther fractionated on a Sephadex gel filtration column equilibratedwith 8M urea in the Tris-HCl buffer. Fractions containing decorin werecollected.

The core protein is purified from cloned cell lines transfected with thepSV2-decorin/CP vector or the vector containing the alanine-mutated cDNAand amplified as described above. These cells are grown to confluency asdescribed above. At confluency the cell monolayer is washed four timeswith serum-free medium and incubated in e MEM supplemented with 2 mMglutamine for 2 hours. This spent medium is discarded. Cells are thenincubated with α MEM supplemented with 2 mM glutamine for 24 hours andthe spent media are collected and immediately made to 0.5 mMphenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 0.04 mg/ml aprotininand 5 mM EDTA as serum-free spent media. The spent media are firstpassed through gelatin-Sepharose and the flow-through fraction is thenbatch-absorbed to CM-Sepharose Fast Flow (Pharmacia Fine Chemicals,Piscataway, N.J.) preequilibrated in 50 mM Tris/HCl, pH 7.4 containing0.1M NaCl. After overnight incubation at 4° C., the slurry is pouredinto a column, washed extensively with the preequilibration buffer andeluted with 0.1M-1M linear gradient of NaCl in 50 mM Tris/HCl, pH 7.4.The fractions containing decorin are pooled, dialyzed against 50 mM NH₄HCO₃ and lyophilized. The lyophilized material is dissolved in 50 mMTris, pH 7.4, containing 8M urea and applied to a Sephacryl S-200 column(1.5×110 cm). Fractions containing decorin core proteins as revealed bySDS-polyacrylamide electrophoresis are collected and represent purifieddecorin core protein.

EXAMPLE II Binding of TGFβ to Decorin

a. Affinity Chromatography of TGFβ on Decorin-Sepharose

Decorin and gelatin were coupled to cyanogen bromide-activated Sepharose(Sigma) by using 1 mg of protein per ml of Sepharose matrix according tothe manufacturer's instructions. Commercially obtained TGFβ-1(Calbiochem, La Jolla, Calif.) was ¹²⁵ I-labelled by the chloramine Tmethod (Frolik et al., J. Biol. Chem. 259:10995-11000 (1984)) which isincorporated herein by reference and the labeled TGFβ was separated fromthe unreacted iodine by gel filtration on Sephadex G-25, equilibratedwith phosphate buffered saline (PBS) containing 0.1% bovine serumalbumin (BSA) (FIG. 2A). [¹²⁵ I]-TGFβ1 (5×10⁵ cpm) was incubated inBSA-coated polypropylene tubes with 0.2 ml of packed decorin-Sepharose(574 ) or gelatin-Sepharose (0) in 2 ml of PBS pH 7.4, containing 1MNaCl and 0.05% Tween 20. After overnight incubation, the affinitymatrices were transferred into BSA-coated disposable columns (Bio Rad)and washed with the binding buffer. Elution was effected first with 3MNaCl in the binding buffer and then with 8M urea in the same buffer.Fractions were collected, counted for radioactivity in a gamma counterand analyzed by SDS-PAGE under nonreducing condition using 12% gels.

FIG. 2A shows the radioactivity profile from the two columns and theSDS-PAGE analysis of the fractions is shown in FIG. 2B. The TGFβ-1starting material contains a major band at 25 kd. This band representsthe native TGFβ-1 dimer. In addition, there are numerous minor bands inthe preparation. About 20-30% of the radioactivity binds to the decorincolumn and elutes with 8M urea, whereas only about 2% of theradioactivity is present in the urea-eluted fraction in the controlfractionation performed on gelatin-Sepharose (FIG. 2A). Thedecorin-Sepharose nonbound fraction contains all of the minor componentsand some of the 25 kd TGFβ-1, whereas the bound, urea-eluted fractioncontains only TGFβ-1 (FIG. 2B). These results show that TGFβ-1 bindsspecifically to decorin, since among the various components present inthe original TGFβ-1 preparation, only TGFβ-1 bound to thedecorin-Sepharose affinity matrix and since there was very littlebinding to the control gelatin-Sepharose affinity matrix. The TGFβ-1that did not bind to the decorin-Sepharose column may have beendenatured by the iodination. Evidence for this possibility was providedby affinity chromatography of unlabeled TGFβ-1 as described below.

In a second experiment, unlabeled TGFβ-1 180 ng was fractionated ondecorin-Sepharose as described above for ¹²⁵ I-TGFβ.

TGFβ-1 (180 ng) was incubated with decorin-Sepharose or BSA-agarose (0.2ml packed volume) in PBS (pH 7.4) containing 1% BSA. After overnightincubation at 4° C., the resins were washed with 15 ml of the buffer andeluted first with 5 ml of 3M NaCl in PBS then with 5 ml of PBScontaining 8M urea. Aliquots of each pool were dialyzed against culturemedium without serum and assayed for the inhibition of [³ H]thymidineincorporation in MvlLu cells (Example III). The amounts of TGFβ-1 ineach pool were calculated from the standard curve of [³ H]thymidineincorporation obtained from a parallel experiment with knownconcentration of TGFβ-1. The results show that the TGFβ-1 boundessentially quantitatively to the decorin column, whereas there waslittle binding to the control column (Table 1). The partial recovery ofthe TGFβ-1 activity may be due to loss of TGFβ-1 in the dialyses.

                  TABLE I                                                         ______________________________________                                        Decorin-Sepharose affinity chromatography of nonlabeled TGFβ-1           monitored by growth inhibition assay in Mv1Lu cells.                                      TGFβ-1 (ng)                                                  Elution       Decorin-Sepharose                                                                           BSA-Sepharose                                     ______________________________________                                        Flow through & wash                                                                         2.7 (2.3%)    82.0 (93.9%)                                      3 M NaCl      2.2 (1.8%)    1.3 (1.5%)                                        8 M Urea      116.0 (95.9%) 4.0 (4.6%)                                        ______________________________________                                    

b. Binding of TGFβ-1 to Decorin in a Microtiter Assay: Inhibition byCore Protein and Byglycan

The binding of TGFβ-1 to decorin was also examined in a microtiterbinding assay. To perform the assay, the wells of a 96-well microtiterplate were coated overnight with 2 μg/ml of recombinant decorin in 0.1Msodium carbonate buffer, pH 9.5. The wells were washed with PBScontaining 0.05% Tween (PBS/Tween) and samples containing 5×10⁴ cpm of[¹²⁵ I]-TGFβ-1 and various concentrations of competitors in PBS/Tweenwere added to each well. The plates were then incubated at 37° C. for 4hours (at 4° C. overnight in experiments with chondroitinaseABC-digested proteoglycans), washed with PBS/Tween and the boundradioactivity was solubilized with 1% SDS in 0.2M NaOH. Total bindingwithout competitors was about 4% under the conditions used. Nonspecificbinding, determined by adding 100-fold molar excess of unlabeled TGFβ-1over the labeled TGFβ-1 to the incubation mixture, was about 13% oftotal binding. This assay was also used to study the ability of otherdecorin preparations and related proteins to compete with theinteraction.

Competition of the decorin binding was examined with the followingproteins (FIGS. 3A and B: symbols are indicated in the section of BRIEFDESCRIPTION OF THE FIGURES):

Decorin isolated from bovine skin and biglycan isolated from bovinearticular cartilage (PGI and PGII, obtained from Dr. Lawrence Rosenberg,Monteflore Medical Center, N.Y.; and described in Rosenberg et al., J.Biol. Chem. 250:6304-6313, (1985), incorporated by reference herein),chicken cartilage proteoglycan (provided by Dr. Paul Goetinck, La JollaCancer Research Foundation, La Jolla, Calif., and described in Goetinck,P. F., in THE GLYCOCONJUGATES, Vol. III, Horwitz, M. I., Editor, pp.197-217, Academic Press, NY). For the preparation of core proteins,proteoglycans were digested with chondroitinase ABC (Seikagaku, Tokyo,Japan) by incubating 500 μg of proteoglycan with 0.8 units ofchondroitinase ABC in 250 μl of 0.1M Tris/Cl, pH 8.0, 30 mM sodiumacetate, 2 mM PMSF, 10 mM N-ethylmalelmide, 10 mM EDTA, and 0.36 mMpepstatin for 1 hour at 37° C. Recombinant decorin and decorin isolatedfrom bovine skin (PGII) inhibited the binding of [¹²⁵ I]-TGFβ-1, asexpected (FIG. 3A). Biglycan isolated from bovine articular cartilagewas as effective an inhibitor as decorin. Since chicken cartilageproteoglycan, which carries many chondroitin sulfate chains, did notshow any inhibition, the effect of decorin and biglycan is unlikely tobe due to glycosaminoglycans. Bovine serum albumin did not shown anyinhibition. This notion was further supported by competition experimentswith the mutated decorin core protein (not shown) and chondroitinaseABC-digested decorin and biglycan (FIG. 3B). Each of these proteins wasinhibitory, whereas cartilage proteoglycan core protein was not. Thedecorin and biglycan core proteins were somewhat more active than theintact proteoglycans. Bovine serum albumin treated with chondroitinaseABC did not shown any inhibition. Additional binding experiments showedthat [¹²⁵ I]-TGFβ-1 bound to microtiter wells coated with biglycan orits chondroitinase-treated core protein. These results show that TGFβ-1binds to the core protein of decorin and biglycan and implicates theleucine-rich repeats these proteins share as the potential bindingsites.

EXAMPLE III Analysis of the Effect of Decorin on Cell ProliferationStimulated or Inhibited by TGFβ-1

The ability of decorin to modulate the activity of TGFβ-1 was examinedin [³ H]thymidine incorporation assays. In one assay, an unamplified CHOcell line transfected only with pSV2dhfr (control cell line A inreference 1, called CHO cells here) was used. The cells were maintainedin nucleoside-free alpha-modified minimal essential medium (α-MEM,GIBCO, Long Island, N.Y.) supplemented with 9% dialyzed fetal calf serum(dFCS) and [³ H]thymidine incorporation was assayed as described(Cheifetz et al., Cell 48:409-415 (1987)). TGFβ-1 was added to the CHOcell cultures at 5 ng/ml. At this concentration, it induced a 50%increase of [³ H]thymidine incorporation in these cells. Decorin or BSAwas added to the medium at different concentrations. The results areshown in FIG. 4A. The data represent percent neutralization of theTGFβ-1-induced growth stimulation, i.e., [³ H]thymidine incorporation,in the absence of either TGFβ-1 or decorin=0%, incorporation in thepresence of TGFβ-1 but not decorin=100%. Each point shows the mean±standard deviation of triplicate samples. Decorin () BSA (0).

Decorin neutralized the growth stimulatory activity of TGFβ-1 with ahalf maximal activity at about 5 μg/ml. Moreover, additional decorinsuppressed the [³ H]-thymidine incorporation below the level observedwithout any added TGFβ-1, demonstrating that decorin also inhibited TGFβmade by the CHO cells themselves. Both the decorin-expressor and controlCHO cells produced an apparently active TGFβ concentration of about 0.25ng/ml concentration into their conditioned media as determined by theinhibition of growth of the mink lung epithelial cells. (The assay couldbe performed without interference from the decorin in the culture mediabecause, as shown below, the effect of TGFβ on the mink cells was notsubstantially inhibited at the decorin concentrations present in thedecorin-producer media.)

Experiments in MvLu mink lung epithelial cells (American Type CultureCollection CCL64) also revealed an effect by decorin on the activity ofTGFβ-1. FIG. 4B shows that in these cells, the growth of which ismeasured by thymidine incorporation, had been suppressed by TGFβ-1.Assay was performed as in FIG. 4A, except that TGFβ-1 was added at 0.5ng/ml. This concentration of TGFβ induces 50% reduction of [³H]-thymidine incorporation in the Mv1Lu cells. The data representneutralization of TGFβ-induced growth inhibition; i.e., [³ H]-thymidineincorporation in the presence of neither TGFβ or decorin=100%;incorporation in the presence of TGFβ but not decorin=0%.

EXAMPLE IV New Decorin-Binding Factor that Controls Cell Spreading andSaturation Density

Analysis of the decorin contained in the overexpressor culture media notonly uncovered the activities of decorin described above, but alsorevealed the presence of other decorin-associated growth regulatoryactivities. The overexpressor media were found to contain a TGFβ-likegrowth inhibitory activity. This was shown by gel filtration of theDEAE-isolated decorin under dissociating conditions. Serum-freeconditioned medium of decorin overexpressor CHO-DG44 cells transfectedwith decorin cDNA was fractionated by DEAE-Sepharose chromatography in aneutral Tris-HCl buffer and fractions containing growth inhibitoryactivity dialyzed against 50 mM NH₄ HCO₃, lyophilized and dissolved in4M with guanidine-HCl in a sodium acetate buffer, pH 5.9. The dissolvedmaterial was fractionated on a 1.5×70 cm Sepharose CL-6B columnequilibrated with the same guanidine-HCl solution. The fractions wereanalyzed by SDS-PAGE, decorin ELISA and cell growth assays, alldescribed above. Three protein peaks were obtained. One contained highmolecular weight proteins such as fibronectin (m.w. 500,000) and nodetectable growth regulatory activities, the second was decorin with theactivities described under Example III and the third was a low molecularweight (10,000-30,000-dalton) fraction that had a growth inhibitoryactivity in the mink cell assay and stimulated the growth of the CHOcells. FIG. 5A summarizes these results. Shown are the ability of thegel filtration fractions to affect [³ H]-thymidine incorporation by theCHO cells and the concentration of decorin as determined by enzymeimmunoassay. Shown also (arrows) are the elution positions of molecularsize markers: BSA, bovine serum albumin (Mr=66,000); CA, carbonicanhydrase (Mr=29,000); Cy, cytochrome c (Mr=12,400); AP, aprotinin(Mr=6,500); TGF, [¹²⁵ I]TGFβ-1 (Mr=25,000).

The nature of the growth regulatory activity detected in the lowmolecular weight fraction was examined with an anti-TGFβ-1 antiserum.The antiserum was prepared against a synthetic peptide from residues78-109 of the human mature TGFβ-1. Antisera raised by others against acyclic form of the same peptide, the terminal cysteine residues of whichwere disulfide-linked, have previously been shown to inhibit the bindingof TGFβ-1 to its receptors (Flanders et al., Biochemistry 27:739-746(1988), incorporated by reference herein). The peptide was synthesizedin an Applied Biosystems solid phase peptide synthesizer and purified byHPLC. A rabbit was immunized subcutaneously with 2 mg per injection ofthe peptide which was mixed with 0.5 mg of methylated BSA (Sigma, St.Louis, Mo.) and emulsified in Freund's complete adjuvant. The injectionswere generally given four weeks apart and the rabbit was bledapproximately one week after the second and every successive injection.The antisera used in this work has a titer (50% binding) of 1:6,000 inradioimmunoassay, bound to TGFβ-1 in immunoblots.

This antiserum was capable of inhibiting the activity of purified TGFβ-1on the CHO cells. Moreover, as shown in FIG. 5B, the antiserum alsoinhibited the growth stimulatory activity of the low molecular weightfraction as determined by the [³ H]-thymidine incorporation assay on theCHO cells. Increasing concentrations of an IgG fraction prepared fromthe anti-TGFβ-1 antiserum suppressed the stimulatory effect of the lowmolecular weight fraction in a concentration-dependent manner (). IgGfrom a normal rabbit serum had no effect in the assay (◯).

The above result identified the stimulatory factor in the low molecularweight fraction as TGFβ-1. However, TGFβ-1 is not the only activecompound in that fraction. Despite the restoration of thymidineincorporation by the anti-TGFβ-1 antibody shown in FIG. 5B, the cellstreated with the low molecular weight fraction were morphologicallydifferent from the cells treated with the control IgG or cells treatedwith antibody alone. This effect was particularly clear when theantibody-treated, low molecular weight fraction was added to cultures ofH-ras transformed NIH 3T3 cells (Der et al., Proc. Natl. Acado Sci. USA79:3637-3640 (1982)). As shown in FIGS. 6A and 6B, cells treated withthe low molecular weight fraction and antibody (micrograph in panel B)appeared more spread and contact inhibited than the control cells(micrograph in panel A). This result shows that the CHO cell-derivedrecombinant decorin is associated with a cell regulatory factor, MRF,distinct from the well characterized TGFβ's.

Additional evidence that the new factor is distinct from TGFβ-1 camefrom HPLC experiments. Further separations of the low molecular weightfrom the Sepharose CL-6B column was done on a Vydac C4 reverse phasecolumn (1×25 cm, 5 μm particle size, the Separations Group, Hesperia,Calif.) in 0.1% trifluoroacetic acid. Bound proteins were eluted with agradient of acetonitrite (22-40%) and the factions were assayed forgrowth-inhibitory activity in the mink lung epithelial cells and MRFactivity in H-ras 3T3 cells. The result showed that the TGFβ-1 activityeluted at the beginning of the gradient, whereas the MRF activity elutedtoward the end of the gradient.

The deposit of the CHO-DG44 cells transfected with pSV2-decorin was madeunder the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure and the Regulations thereunder (Budapest Treaty). This assuresmaintenance of a viable culture for 30 years from date of deposit. Thedeposits will be made available by ATCC under the terms of the BudapestTreaty, and subject to an agreement between Applicants and ATCC whichassures permanent and unrestricted availability upon issuance of thepertinent U.S. patent. The Assignee herein agrees that if the culture ondeposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced upon notification witha viable specimen of the same culture. Availability of the deposits isnot to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The deposit was made for the convenience of the relevant public and doesnot constitute an admission that a written description would not besufficient to permit practice of the invention to the specificconstruct. Set forth hereinabove is a complete written descriptionenabling a practitioner of ordinary skill to duplicate the constructdeposited and to construct alternative forms of DNA, or organismscontaining it, which permit practice of the invention as claimed.

Although the invention has been described with reference to thepresently-preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

What is claimed is:
 1. A method of inhibiting an activity of TGFβ,comprising contacting TGFβ with an effective amount of purified decorin.2. The method of claim 1, wherein the TGFβ is TGFβ-1.
 3. The method ofclaim 1, wherein the TGFβ is TGFβ-2.
 4. The method of claim 1, whereinthe inhibited activity is promotion of cell proliferation.
 5. The methodof claim 1, wherein the inhibited activity is suppression of cellproliferation.
 6. The method of claim 1, wherein the activity ispromotion of extracellular matrix production.